The proposed research will evaluate relative cardiovascular toxicities of diverse, inhaled tobacco products, using an innovative physiological assay developed in the Principal Investigator's laboratory, in response to FOA PAR-12-267, Tobacco Control Regulatory Research. The results of this unique and innovative approach will inform the FDA about how different types of cigarettes and related smoked tobacco products vary in their harmful effects on the endothelial lining of blood vessels, an important contributor o cardio-vascular disease. The research will also yield valuable pre-market toxicity screens for new and emerging tobacco products. This proposal uses a recently-developed micro-ultrasound-based approach to measure endothelial function in living rats in the form of flow-mediated vasodilation (FMD), which is impaired in humans by both active smoking and secondhand smoke exposure. This approach has enabled detection of acute cardiovascular toxicity resulting from brief exposure to tobacco smoke at real-world levels. Rats have advantages over humans including minimal inter-subject variability and the ability to harvest and analyze tissue, and the FMD measurement approach is more physiologically relevant than classic ex vivo studies of isolated segments of aorta. Therefore, this approach can assess relative toxicity of exposure to different tobacco products under physiological conditions without the confounding variables of genetics and lifestyle that are inherent to human studies. Rats can also be exposed to purified constituents of smoke that are suspected mediators of endothelial dysfunction, and to antagonists of those constituents, enabling determination of whether these constituents exert deleterious cardiovascular effects and thus can be used to evaluate toxicity of emerging products. The project will determine extent of impairment of FMD, as well as dose response and short-term recovery, and will illuminate differences in effects of post-exposure plasma on endothelial cells ex vivo and in vivo. Aim 1 is to measure differences in vascular toxicity between different types of smoked tobacco products (consumer and reference cigarettes with varying amounts of nicotine and menthol, cigarillos, and little cigars). This provides information about relative extents and causes of toxicity from different smoked tobacco products, and establishes a solid scientific basis to inform whether cigarillos and little cigars should be regulated like cigarettes. Aim 2 is to determine involvement of specific smoke components in acute endothelial toxicity. This establishes whether specific smoke constituents are involved in smoke-induced endothelial dysfunction and can be used to evaluate toxicity of emerging tobacco products. Aim 3 is to evaluate and understand cardiovascular toxicity of vapors from e-cigarette of varying nicotine levels and vehicle chemical composition. This provides crucial cardiovascular toxicity assessment of these inhaled nicotine-delivery vapors relative to tobacco smoke inhalation, and establishes a solid scientific basis for regulatory decisions regarding e-cigarettes.